Tang Liangjie, Hopper Erin D, Tong Yan, Sadowsky Jack D, Peterson Kimberly J, Gellman Samuel H, Fitzgerald Michael C
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Anal Chem. 2007 Aug 1;79(15):5869-77. doi: 10.1021/ac0700777. Epub 2007 Jun 21.
The equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy (DeltaGf) and m-value (deltaDeltaGf/delta[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (> approximately 15 kDa) or multidomain proteins. In the absence of ligand, DeltaGf and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein-ligand dissociation constants (Kd values) could also be ascertained. The SUPREX-derived binding free energies and Kd values evaluated here were in good agreement with those reported on the same complexes using other techniques.
利用SUPREX（基于氢/氘交换速率的未纯化蛋白质稳定性）对四种模型蛋白系统的平衡去折叠特性进行了表征。SUPREX是一种基于氢/氘交换和质谱的技术，用于测量与蛋白质折叠/去折叠反应相关的自由能（ΔGf）和m值（δΔGf/δ[变性剂]）。本研究中的模型蛋白（钙调蛋白、碳酸酐酶II、RmlB、Bcl-xL）被选来测试SUPREX在更大（>约15 kDa）或多结构域蛋白热力学分析中的适用性。在没有配体的情况下，无法使用先前为SUPREX建立的传统数据采集和分析方法来评估这些蛋白的ΔGf和m值。然而，蛋白的配体结合形式适用于传统的SUPREX分析，并且有可能评估所选配体相当准确和精确的结合自由能。在某些情况下，还可以确定蛋白-配体解离常数（Kd值）。此处评估的源自SUPREX的结合自由能和Kd值与使用其他技术报道的相同复合物的结果高度一致。
